Caucasian Mutation Research Articles

Abstract 1906: MSH3: An underestimated DNA MMR gene in colorectal carcinogenesis and its potential role in disparity

Abstract Background: Unique mutations in cancer genes have been found in different races including African Americans (AA) population. Maintenance of proficient mismatch repair genes is important in colorectal cancer. Among them, MSH3 is found to be more important in colorectal cancer in AA particularly as its alterations associate with EMAST phenotype that has poor prognosis. However, there are regional variations in the classification and criteria used to determine the pathogenicity of these mutations particularly in AA due to their high genomic diversity. Aim: To analyze unique and novel MSH3 mutations using specific in-silico prediction tools and to validate their biological significances in AA-CRC. Results: In this study, we validated our targeted exome sequencing data of MSH3 mutations in AA and Caucasians (CA) CRC samples. A cost effective in-silico approach was employed to narrow down most significant MSH3 mutations. Interestingly, we and found six non-synonymous novel, pathogenic mutations in MSH3 gene (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, c.G3241A) correspond to MSH3 protein (E413K; S466N; S920F; E976K; H1010Y; E1081K), in AA than CA samples. Also, we observed loss or gain of different chemical bonding patterns (hydrogen, ionic, hydrophobic, and di-sulfide bonding) between wild type and mutant MSH3 protein. Interestingly, some of these mutations were within the ATPase site while others are in MSH3-MSH2 interacting domains. Furthermore, Molecular Dynamics Simulation (MDS) of these six mutants of MSH3 proteins showed an overall effect on MSH3 protein stability especially in MSH3-MSH2 interaction compared to wild type MSH3 protein.Conclusion: Our data showed that, six MSH3 non-synonymous mutations are deleterious as they affect important sites within MSH3-MSH2 interactive and ATPase domain. Assessing the real frequency of these mutations in large cohort and their specific effect on function may shed light on EMAST phenotype, high prevalence, and overall poor prognosis in the AA population. In vitro functional assays using CRISPR-Cas9 knock-in and in vivo studies are underway to mimic these mutations. Citation Format: Mudasir Rashid, Rumaisa Rashid, Minoru Koi, John M. Carethers, Hassan Brim, Hassan Ashktorab. MSH3: An underestimated DNA MMR gene in colorectal carcinogenesis and its potential role in disparity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1906.

Therapeutic effects of drugs developed for CFTR mutations in Caucasians on a Japanese Q98R CFTR mutation

Therapeutic effects of drugs developed for CFTR mutations in Caucasians on a Japanese Q98R CFTR mutation

DIFFERENCES IN THE FREQUENCY OF POLYMORPHISMS IN THE HFE GENE (H63D AND C282Y) IN A POPULATION GROUP FROM NORTHWEST SÃO PAULO, BRAZIL

Hereditary hemochromatosis (HH) is an autosomal recessive disease characterized by excess iron. People with polymorphisms in the HFE gene ( H63D and C282Y ) may develop inflammatory processes resulting from changes in iron metabolism. The ability of the HFE protein to make connections with transferrin receptors, located on cell membranes, allows it to help control the absorption of iron in the body. This work sought to investigate the frequency of HFE gene variants in a sample from the city of São José do Rio Preto, SP, Brazil. DNA samples from 105 people were subjected to molecular analysis to confirm mutations in the HFE gene ( H63D and C282Y ), using PCR-RFLP. The group was separated by male and female gender, age group of 10 in 10 years, and ethnic groups. Statistical analyzes were performed using the IBM SPSS Statistics 20 software and data were presented as percentages, with a significance level of 0.05. The associations between the wild-type homozygote, heterozygote, and mutant homozygote genotypes for each mutation were tested by the Chi-Square test. Then, the allele frequencies in the genotypes were calculated. Fisher's exact test was performed to verify the association between gender, age group, and ethnicity with mutations in the HFE gene. The sample included: 55 men (52.38%) and 50 women (47.62%), with ages ranging from 18 to 62 years, with an average of 30 years. A total of 210 alleles were evaluated and for the H63D mutation, 78 wild-type homozygotes, 26 heterozygotes, and 1 mutant homozygote were found. For the C282Y mutation, 99 wild-type homozygotes, 6 heterozygotes and no mutant homozygote were found. Allelic frequency analysis indicated the occurrence of significant differences between wild-type homozygous, heterozygous, and mutant homozygous genotypes for the mutations (p < 0.05). Fisher's exact test indicated no association between gender, age, and ethnicity for the H63D and C282Y polymorphisms (p > 0.05). The heterozygous genotype for H63D was more frequent in men (15-14%) than women 11 (10%). For C282Y equal frequencies (3-3%) were found for heterozygosity between men and women. H63D heterozygosity was more frequent in Caucasians (22-21%). In African-derived, only one individual was heterozygous for H63D . The frequency of HH genotypes varies among different populations. The low incidence or absence of homozygous alleles is due to the different degrees of miscegenation in the formation of the Brazilian population. The H63D mutation is an important marker for the Brazilian population, being frequent in Africa, although it has low phenotypic expression. The C282Y is predominantly associated with homozygosity for clinical cases, however, it is rare in African populations and frequent in Caucasians. Our results corroborate the data in the literature, with a predominance of the H63D mutation in Caucasians, African-derived, and Afro-descendants, and C282Y in Caucasians. The frequency of H63D and C282Y polymorphisms in the HFE gene showed a higher occurrence of heterozygotes for H63D in this population group. Studies to characterize HH are needed to devise strategies for effective support to patients and, therefore, improve quality of life.

P–531 hCFTR p.G970D mutation causes Sertoli Cell-only Syndrome (SCOS) and Congenital bilateral absence of the vas deferens (CBAVD)

Abstract Study question Whether CFTR is a pathogenic gene for azoospermia? Summary answer CFTR p.G970D affects spermatogenesis and leads to male infertility by affecting the proliferation and survival of Germ Cell. What is known already Male infertility is a multifactorial and heterogeneous pathological condition affecting 7% of the general male population. However, up to now, only a relatively low number of genic factors have a clear relationship with spermatogenesis. Although, increased frequency of CFTR mutations or impaired CFTR expression in men with non-obstructive azoospermia or oligospermia as compared to the fertile men has been reported, but there is no direct evidence CFTR mutations cause azoospermia. Compared to F508Del mutations in Caucasians, p.G970D mutation is the most frequent CFTR mutation identified in Chinese CF patients. However, p.G970D has not been reported involved with male infertility. Study design, size, duration In this study, began in an infertile man suffering CBAVD and SCOS with no CF-like phenotype related symptoms up to now. By identifying the patient with CFTR p.G970D mutation, we further verified the function of the mutation in spermatogenesis in spermatogonia cell lines. Control testicular tissue sample was obtained from fertility man donors. Participants/materials, setting, methods WES was performed for probands and relatives and the mutation was confirmed by Sanger sequencing. Hematoxylin-eosin (HE) staining and immune fluorescence (IF) was performed on seminiferous tubules from the patient and control to characterize the structural anomalies present in the patient. GC2 mCFTRG965D cells was knocked in by the CRISPR/Cas9 gene editing system. The effects of mutations on the growth and proliferation of GC2 cells were detected by CCK8, IF, WB, BCECF staining and RT-PCR. Main results and the role of chance First, we identified the CBAVD and SCOS patient with homozygous missense mutations p.G970D in the CFTR gene, and his mutation inherited from both parents. The patient has normal general parameters and fertility parameters except for smaller testes, lower semen volume and pH. His testicular histopathology and co-location of CFTR and DDX4 which is the marker of spermatogonia likewise showed SCOS. Second. given that the amino acid sequence is conserved and the same expression and localization patterns of CFTR between human and mouse, we generated mouse derived cell lines model (mCFTRG965D) that carried a homozygous mutation equivalent to the CFTR variant in patients, using CRISPR/Cas9-mediated genome editing. mCFTRG965D affects the proliferation of Germ Cell, but has less effect on Sertoli cells, which is similar to the SCOS patient’s phenotype. Third, lower mature CFTR were observed in the GC2 mCFTRG965D groups cells compared to those in wild type groups, and CFTR protein is not evident in the GC2 mCFTRG965D groups’ cell membrane, which demonstrated the mutation affecting the anchoring of CFTR to the cell membrane. What’s more, the missense mutation will affect the function of CFTR in regulating pH, thus affecting cell homeostasis. Limitations, reasons for caution The low number of biological samples, we need more patients to confirm this mutation and azoospermia. We only validated at the cellular level, not in an animal model. It is noteworthy that, the CFTRF508del mice are fertility. Wider implications of the findings: Our study reveals that CFTR has a broader indication than just the absence of the vas deferens. We recommend to take further understanding of CFTR playing important role in spermatogenesis by affecting germ cell survival not just regulating cell volume during spermiogenesis. Trial registration number Not applicable

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood.We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC.A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation.We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery.

Novel Characteristics of Race-Specific Genetic Functions in Korean CADASIL.

Background and Objectives: Previous studies found differences in the characteristics of NOTCH3 mutations in Caucasians and Asians with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Therefore, we sought to investigate the correlations between genetic and clinical/radiological findings in Korean CADASIL patients including some variants of unknown significance (VUS). Materials and Methods: We screened 198 patients with a suspected diagnosis of CADASIL between 2005 and 2015 via Sanger sequencing. Results: A total of 34 subjects (52.5 ± 9.5 years) were included. The majority of the mutations were in exon 3 and exon 11. R75P mutations (n = 5), followed by Y465C and R544C mutations (n = 4) were the most prevalent. Patients with those mutations exhibited less frequent anterior temporal (AT) or external capsular (EC) hyperintensities compared to patients with other locus mutations. Hemorrhagic stroke (HS) was found to be associated with mutations in exon 3 (R75P), exon 9 (Y465C), exon 11 (R587C), and exon 22 (R1175W variants), which were common locations in our study. Although it is unclear that genetic differences might affect the phenotypes in ethnicities, Asian population shows less migraine or seizure, but more intracerebral hemorrhage. Unlike in westernized countries, typical AT or EC hyperintensities may not be significant MRI markers, at least in Korean CADASIL patients. Furthermore, similar to R75P phenotypes, it is a novel finding that patients with Y465C and R1175W VUS have less frequent AT involvement than Caucasians. Conclusion: The associations between HS and common genetic locations account for the increased development of intracerebral hemorrhage in Koreans rather than Caucasians. We suggest that some CADASIL mutations appear to impart novel region-specific characteristics.

Detection and Differential Diagnosis of Prekallikrein Deficiency: Genetic Study of New Families and Systematic Review of the Literature

Introduction. Prekallikrein (PK) and high-molecular-weight kininogen (HK) deficiencies are ultra-rare, autosomal-recessive defects of the contact system caused by biallelic mutations in the KLKB1 and KNG1 genes, respectively. Since affected subjects do not manifest a bleeding phenotype, a correct diagnosis is essential to prevent the administration of prohemostatic agents or plasma and to avoid delay of surgery.We describe a new case of PK deficiency identified at UMC Mainz. In addition, we performed a systematic review of the literature in order to i) collect blood material for genetic studies of reported PK deficient cases lacking this information, and ii) perform a comprehensive individual patient analysis for studying the clinical course and diagnostic criteria (analysis ongoing).Methods. MEDLINE and EMBASE were searched without time and language restrictions. Reference lists of retrieved articles, abstract books of hematology congresses, theses, and grey literature were manually reviewed. All the authors of recent articles on PK deficient cases not assessed for genetic defects, were contacted in order to retrieve blood samples. Clotting activities of PK and HK (PK:C and HK:C) were measured using PK and HK deficient plasmas, respectively. PK and HK antigens (PK:Ag and HK:Ag) were determined by ELISA. Routine aPTT and coagulation factor measurements were performed using an ACL TOP (IL). Genetic testing was performed by Sanger-, Pyrosequencing and/or NGS.Results. Our patient was a 69-year-old woman of African descent referred for preoperative evaluation of an isolated aPTT prolongation. Diagnosis of PK deficiency was based on absent PK:C and PK:Ag (≤1% of normal, each). The homozygous KLKB1 mutation p.Ser151Phefs*34 was found, which was not yet described as a cause of PK deficiency, but had been detected in the African sub-collective (MAF 1.3%) of the 1000 Genomes cohort.A total of 1,913 studies were identified by systematic literature review. Eleven studies with genetic data were found. Blood from 4 unrelated European families without previous genetic testing was analysed, including 3 index cases and 5 relatives. The KLKB1 mutation p.Cys548Tyr was found in 2 families with one index patient being homozygous. This data and the literature suggest that p.Cys548Tyr may be the most frequent KLKB1 mutation in Caucasians, associated with lacking PK:C but low amount of PK:Ag.One patient erroneously diagnosed with PK deficiency based on (incomplete) normalization of aPTT with increased preincubation time and low PK:C (7%) was found to carry compound heterozygous mutations in KNG1 (c.1038+1G>A and c.1165C>T, p.Arg389*) but no mutations in KLKB1. His low PK:C was explained by severe HK deficiency.Conclusions. PK deficiency may not be as rare as previously thought, especially in subjects of African origin. Incomplete normalization of severely prolonged aPTT upon prolonged preincubation and low PK levels are not sufficient for the diagnosis of PK deficiency. Our latter case and data from literature suggest that patients with HK deficiency usually present with moderately low PK levels: therefore, PK:C, PK:Ag, HK:C, and HK:Ag should be determined for proper diagnosis. DisclosuresKremer Hovinga:Shire: Other: Member of Advisory Board, Research Funding; Ablynx: Other: Member of Advisory Board. Lammle:Siemens: Honoraria, Other: congress travel and accomodation support ; Bayer: Honoraria, Other: congress travel and accomodation support; Alexion: Other: congress travel and accomodation support ; Baxter: Other: congress travel and accomodation support ; Ablynx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: congress travel and accomodation support .

P2.03b-009 Brain Metastasis and Epidermal Growth Factor Receptor Mutations in Croatian Caucasians with Lung Adenocarcinoma: Topic: Brain Meta

The brain is a common site of metastasis in non-small cell lung cancer (NSCLC). The aim of this study was two-fold: 1) to determine the incidence of brain metastasis (BM) in Caucasian lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutations and 2) to evaluate the frequencies and potential relationship of the different EGFR mutations with BM. A retrospective cohort study was conducted at a Croatian tertiary hospital (Clinic for Respiratory Diseases “Jordanovac”) using data collected from medical records. Caucasian patients with primary NSCLC who were tested for EGFR mutation status between January 2014 and October 2015 were included. Of 1040 NSCLC samples tested, 122 (11.7%) patients with lung adenocarcinoma harbored EGFR mutations; six EGFR positive (+) patients (four with BM) had repeat EGFR testing. The majority of EGFR mutants were females (n= 90, 77.6%), non-smokers (including never-smokers and former-smokers; n= 95, 92.2%), diagnosed with advanced disease (stage IIIB/IV) at first presentation (n= 75, 68.8%), and median age at initial diagnosis of primary lung cancer was 65 years (35 - 90). Twenty-three (19.8%) of 116 EGFR+ patients were diagnosed with BM; for six EGFR+ patients, data about BM was missing. Most were 64 years of age or younger (n= 15, 65.2%) at diagnosis of BM (median age: 62 years, 48 - 72). Synchronous BM disease at initial diagnosis of lung cancer was found in 43.5% of EGFR+ patients with BM (n= 10). There were more EGFR+ women with BM (n= 20, 87%) than men. Single exon 19 deletion and exon 21 L858R mutations were the most common subtypes in both EGFR+ patients without BM (n= 44, 47.3% and n= 27, 27.8%, respectively) and with BM (n= 13, 56.5% and n= 5, 21.7%, respectively). One BM patient (4.3%) had a double mutation (exon 19 and 21), while six non-BM patients (6.2%) had simultaneous pairings, most commonly between exon 19 and 20 (n=3, 3.1%). Although exon 18 mutations were seen in six patients without BM (6.2%), none were found in BM+EGFR+ patients. Exon 20 T790M mutation occurred in 17.4% of BM patients (n= 4) versus 15.3% of non-BM patients (n= 15). Rare EGFR double mutations (exon 18 and 20) were found in two non-BM patients (2.2%). Larger long-term prospective studies to explore and confirm these results in BM+EGFR+ patients are warranted. In the era of precision oncology, molecular testing of EGFR mutations may further clarify the pathogenesis of lung cancer-associated BM.

An analysis of outcomes in children with cystic fibrosis in a tertiary African centre: A retrospective study.

&lt;p&gt;&lt;strong&gt;Abstract&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; Cystic Fibrosis (CF) is a common genetic disorder in Caucasians that has become increasingly prevalent in populations of African descent. The clinical presentation in African children with CF is commonly related to nutritional and growth abnormalities.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Aims/Objectives&lt;/strong&gt;: To describe clinical, laboratory and spirometric characteristics of children followed up at the CF Clinic at Inkosi Albert Luthuli Central Hospital, Durban, South Africa.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods&lt;/strong&gt;: A retrospective chart review of clinical, laboratory and spirometric data of patients registered from January 2013 to January 2016.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; The data was reviewed for 15 patients mean age 132 months (range 26- 219 months), with 53% males. 60% of these children were Caucasians, 26,7% of these being of black African descent. Collectively, the mean age of diagnosis was 45 months (0- 156), although this was higher in the non-Caucasians at 104 months (range 48-156) versus 1,3 months (range 0-3) in Caucasians. The Caucasian group had better nutritional status when compared to non-Caucasians with BMI of 17,2kg/m&lt;sup&gt;2&lt;/sup&gt; vs 14,5 kg/m&lt;sup&gt;2&lt;/sup&gt;, respectively. Age at diagnosis had a negative correlation with weight-for-age &lt;em&gt;Z &lt;/em&gt;score (-0,61; p &amp;lt; 0.05) and BMI (-0,54; p&amp;lt;0.05). The mean FEV&lt;sub&gt;1&lt;/sub&gt; % predicted was 70.0 (16,1- 120,2). FEV&lt;sub&gt;1&lt;/sub&gt;% predicted had a positive correlation with both weight z-score (0,83; p &amp;lt; 0.001) and BMI (0.59; p &amp;lt; 0.05). Chronic pseudomonas infection occurred only in 2 patients, both of whom were above the age of 16 years. On mutational analysis 5 of the non-Caucasian patients had no mutations identified on the 30 panel mutation used for testing. phelF508.del was the most commonly identified mutation in Caucasians; with 4 homozygotes and 4 heterozygotes.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;: Cystic fibrosis is diagnosed late in non-Caucasian children in South Africa impacting their growth and lung functions. There is a need for a genetic panel that includes mutations specific for children of African descent.&lt;/p&gt;

Mutation spectrum of Egyptian children with cystic fibrosis.

ObjectiveTo know the common CFTR mutations in the Egyptian patients with cystic fibrosis as it was previously thought to be uncommon disease in Egypt.MethodsThis is a cross sectional study of 60 patients diagnosed as cystic fibrosis by sweat chloride testing. They were enrolled from the Allergy and Pulmonology Unit Children’s Hospital Cairo University. They were screened for the presence of the frequent 36 mutations in Caucasians by reverse hybridization line probe technique, using INNO-LiPACFTR19 and CFTR17 + Tn kits.ResultsMost of patients presented with classic manifestations of CF such as variable pulmonary disease and pancreatic insufficiency, and hepatomegaly with or without ascites. The mutations detected were F508 del (58 %), 2183AA/G (10 %), N1303K (6 %), I148T (4 %), W1282X (4 %), G155D (2 %), CFTRdel2-3 (21 KB) (2 %), 3199del6 (2 %), R347P (2 %). Unique to the Egyptian population are these mutations R1162X and A544E (6, 4 %) respectively they were found in our cohort study and were not reported elsewhere in the Arab population till now. There was no association between the initial clinical presentation of CF and the genotypes detected.ConclusionThe F508 del is still the most commonly encountered mutation (58 %), however other rare mutations were identified where each ranged from (2 to 10 %).

P.G970D is the most frequent CFTR mutation in Chinese patients with cystic fibrosis.

Cystic fibrosis (CF), the most common life-threatening autosomal recessive disorder in Caucasians, is caused by mutations in CF transmembrane conductance regulator gene (CFTR). We and others previously identified CFTR mutations in 20 Chinese patients with CF. In this study, eight Chinese patients with a clinical diagnosis of suspected CF were newly collected and screened for CFTR mutations using a combination of conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis. The CFTR mutations observed in Chinese CF patients, both reported previously and identified in the present study, were also summarized. In the newly collected patients, we identified 10 different CFTR mutations, including p.F508del, the most common CF-causing mutation in Caucasians, and three novel mutations (p.V1212Afs*15; p.L666* and p.A969A). Most notably, the previously reported p.G970D mutation was found in six patients, making it the most frequent CFTR mutation identified in Chinese CF patients thus far. In conclusion, we detected p.F508del for the first time, identified additional novel CFTR mutations and recorded the most frequent CF-causing mutation in Chinese CF patients.

Significance of ACADM mutations identified through newborn screening of MCAD deficiency in Japan.

Since the first case was detected in 2000, there has been a remarkable increase in Japanese patients diagnosed with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. Genetic analysis has revealed a spectrum of mutations that is quite different from those observed in Caucasian populations. In 2014, Japan initiated nationwide newborn screening (NBS) for MCAD using tandem mass spectrometry (MS/MS). It is an urgent issue to assess the risk of acute metabolic decompensation from the respective novel mutations found thus far. To evaluate the pathogenic effect of each mutation, we established a eukaryotic cell expression system and prepared 11 mutant proteins identified in five symptomatic patients and eight MS/MS-NBS-positive newborns, as well as two common Caucasian mutations, p.K329E (c.985G>A) and p.Y67H (c.157C>T) for comparison. The expression of four mutant proteins (p.Q45R, p.P92L, p.P128X and p.Y397N) were severely impaired, whereas the others expressed normally, as did p.K329E and p.Y67H. Based on their dehydrogenase activities toward n-octanoyl-CoA, we determined three mutations (p.R53C, p.R281S and p.G362E) to be disease-causing, two mutations having (p.R17H and p.M274V) to be of marginal risk, and two mutations (p.K271E and p.I416T) as benign. Their allele-specific activities were as a whole in accordance with those estimated from the results of measurement in peripheral blood mononuclear cells. As most of the mutations detected in the Japanese population are unique, prudent genetic and enzymatic analysis is essential to precisely evaluate the latent risk of clinical onset for screening-positive newborns.

16 * Ethnicity and phenotype in the SCN5A E1784K mutation

Background: Long QT (LQTS) and Brugada syndromes (BrS) are inherited arrhythmia syndromes characterised by risk of sudden cardiac death (SCD) and highly variable penetrance. Mutations in SCN5A, which encodes the Nav1.5 cardiac ion channel responsible for the inward cardiac sodium current, are responsible for 5-10% of LQTS and associated with 20% of BrS. The E1784K mutation in SCN5A is the most commonly identified SCN5A mutation in both LQTS and BrS, and causes the Long QT type 3 (LQT3)/BrS overlap disease with variable phenotypic expression. Carriers within the same pedigree may exhibit LQT3 phenotype, BrS phenotype or both. The aim of this international project was to determine if there are any associations between phenotype and ethnicity within a unique cohort of E1784K mutation carriers. Methods: 93 E1784K mutation carriers belonging to 33 families from 6 different countries including Japan were included. Data was collected on age, gender, symptoms, family history and the ECG. Non-digital and digital ECG data were analysed using proprietary software. Automatic readings and measurements from 2 observers were taken. A 3rd observer reviewed the ECGs where there was inter-observer discrepancy between the measurements. Statistical significance was determined using either the 2-tailed t-test or Chi-squared test as appropriate. Results: QTc intervals were significantly longer amongst Japanese E1784K mutation carriers, who were more symptomatic albeit with a lower incidence of SCD. Results are summarised in Table [1][1]. The spontaneous type 1 BrS ECG pattern also appeared to be more prevalent in the Japanese compared to Caucasian mutation carriers although the numbers were small. Mean QRS duration was longer amongst Caucasian mutation carriers. Drug-induced BrS phenotype was unmasked in 63% of Caucasians (34/54) who underwent a test. A family history of SCD was present in 50% of Caucasian families compared to only 13.6% of Japanese families (p=0.015). | | Total (n=93) | Caucasian (n=64) | Japanese (n=29) | p-value | | ------------------------- | ------------ | ---------------- | --------------- | ------- | | Age (years, mean, range) | 28, 1-67 | 30, 1-67 | 23, 6-62 | 0.0826 | | Sex-male (n,%) | 47, 51 | 32, 50 | 15, 52 | 0.8776 | | Spontaneous BrS (n,%) | 4, 4 | 1, 2 | 3, 10 | 0.0531 | | QTc (ms, mean, range) | 480, 404-579 | 472, 404-533 | 499, 430-579 | 0.0001 | | PR (ms, mean, range) | 167, 116-302 | 167, 116-302 | 166, 149-180 | 0.8952 | | QRS (ms, mean, range) | 96, 60-139 | 99, 68-139 | 90, 60-120 | 0.0145 | | Pre-syncope/syncope (n,%) | 17, 18 | 8, 13 | 9, 31 | 0.0741 | | Aborted SCD (n,%) | 6, 6 | 6, 9 | 0, 0 | 0.0882 | Table 1 Clinical characteristics Conclusions: There is variation in expression of E1784K between ethnicities with ethnic differences in severity and risk of sudden death. These results support a strong role for genetic modifiers of phenotype and risk which deserves further study. [1]: #T1

Novel Mutation in the CFTR Gene of Cystic Fibrosis Patients in Oman

Introduction: Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder among Caucasians (1: 3,000). In CF, the CFTR gene is frequently mutated, with ΔF508del being the largely common mutation in Caucasians. Our preliminary pilot study in Omani population revealed a CF prevalence of 1:2,738. Objective: The objective of the present study was to determine the most common CFTR mutations in the Omani patients to establish a proper molecular genetics diagnostic basis of CF in Oman. Methods: Blood Genomic DNA samples from Omani patients were examined by PCR and sequencing analyses for the entire coding sequence of the CFTR gene were performed. Results: The innovative aspect of this study was the identification of a novel CF-causing mutation, L578delTA. Furthermore, in contrast to the west, p.S549R appears to be the most common mutation in Oman (65.2% for S549R and 13% for ΔF508). Conclusion: The mutation spectrum of CF in Oman revealed six CF-causing mutations, p.S549R, ΔF508, 3120+1G>A, L578delTA, p.A357T and 3849+10kbC->T. These findings will ultimately pave the way towards the development of molecular genetic tests in Oman to confirm the diagnosis of CF and serve the patient care and management.

A Blistering Case of Hereditary Hemochromatosis

Case Presentation: A 59-year-old male with history of psoriasis and moderate alcohol use presents with blistering of the skin. Biopsies revealed subepidermal bulla. Serum porphyrins were ordered to evaluate for porphyria cutanea tarda (PCT). Total porphyrins were high, 29.1, predominantly uroporphyrin 13.6, consistent with PCT. The patient was also found to have elevated liver related tests ALT 79, AST 53, and serum ferritin of 715. He was referred for further evaluation, and he was found to have elevated transferrin saturation. Genetic testing revealed he was homozygous for the C282Y HFE mutation consistent with hereditary hemochromatosis. Literature Review: Hereditary hemochromatosis (HH) is the most commonly identified genetic mutation in Caucasians, with a prevalence of approximately 1 per 220-250 individuals of northern European origin. The pathophysiologic predisposition to increased inappropriate absorption of dietary iron may lead to development of life-threatening complications of cirrhosis, hepatocellular carcinoma, diabetes, and cardiac disease. Genetic research has led to the discovery of the HFE gene, most notably the C282Y mutation causing hereditary hemochromotosis. A genetic diagnosis can be applied to individuals who have not yet developed any phenotypic expression. These individuals have a genetic susceptibility to develop iron overload, but may or may not do so to a variable degree. Clinical Significance: In this case, the patient presented with a clinical syndrome, (PCT), which, pathophysioligically, has been associated with elevated iron stores. Notably, the patient had a history of being a frequent blood donor, keeping iron levels from rising for most of his life. Mild liver-related lab abnormalities at the time of diagnosis of PCT led to a clinical suspicion of HH. Research Questions: Will early detection of a genetic mutation assist in prevention of complications from this disease? How does PCT relate to HH?

Identification of a SNP in a Regulatory Region of GJB2 Associated With Idiopathic Nonsyndromic Autosomal Recessive Hearing Loss in a Multicenter Study

Additional genetic changes in the regulatory region of the human GJB2 gene encoding the gap junction protein (Connexin 26) may contribute to sensorineural hearing loss. Mutations in GJB2 cause up to 50% of autosomal recessive nonsyndromic hearing impairment (NSHI). In the present study, we screened the putative 5' GJB2 regulatory region for novel alterations. In idiopathic familial cases of NSHI lacking known pathogenic alterations in GJB2, we identified a T→C transition (refSNP: rs117685390) in a putative transcription factor binding sequence 228 bp proximal to the transcriptional start site at a homozygous frequency of 0.125 (n = 40), significantly overrepresented in comparison to the homozygous allele frequencies of 0.043 in the normal-hearing Caucasian population (n = 211; p < 0.001). In a NSHI family, inheritance of the rs117685390 C allele segregated on independent chromosomes with NSHI in conjunction with heterozygous inheritance of c.35delG, the most common Caucasian mutation in the GJB2 coding region. In a patient group (n = 32) bearing heterozygous pathogenic c.35delG mutations, - rs117685390 C allele homozygosity was also highly overrepresented (0.25; p < 0.001) and not exclusively linked to the c.35delG mutation in cis in patients homozygous for c.35delG. However, in the majority of NSHI homozygous c.35delG chromosomes examined (91/94), c.35delG homozygosity was linked to the rs117685390 C allele in cis. These results suggest that the rs117685390 C allele could represent a biomarker for the development of NSHI in Caucasian populations and may be included in risk assessment for the development of NSHI.

Abstract 3063: MicroRNA-605: A novel genetic modifier in Li-Fraumeni Syndrome.

Abstract Background: Li-Fraumeni syndrome (LFS) is a rare autosomal-dominant disorder, typically involving germ-line mutations in the prototypical tumour suppressor gene, TP53. Despite the high penetrance of TP53 mutations, LFS patients display striking differences in the age of disease onset, suggesting the presence of additional genetic modifiers. To date, functional polymorphisms in TP53 (Arg72Pro), MDM2 (SNP 309), as well as DNA copy number variations (CNVs) and telomere attrition, have been associated with cancer risk in LFS. However, it is likely that additional modifiers exist and that a combination of these low-penetrance risk factors act together to predispose TP53 mutation carriers to a spectrum of susceptibilities. Recently, the TP53 signalling network has been linked to a small number of non-coding RNAs, called microRNAs (miRNAs), whose main function is to negatively regulate gene expression. One particular member of this family, called miR-605, has been found to target the MDM2 gene, an important inhibitor of p53 function. Importantly, a common Single Nucleotide Polymorphism (SNP) within miR-605 was also found to modulate gastrointestinal cancer risk. By affecting miR-605 expression, this SNP could impact cancer risk in TP53 mutation carriers. Hypothesis: In this study, we hypothesized that SNP-mediated miR-605 deregulation might modify cancer risk in LFS by eliciting important transcriptional changes in the levels of p53 and/or MDM2. Methods: miR-605 was overexpressed in two TP53 mutant cell lines (RD and Rh30) in order to assess its tumor suppressive functions. MDM2 and TP53 protein expression were analyzed through Western Blotting. Additionally, blood-derived DNA samples from 47 Caucasian TP53 mutation carriers were genotyped for the miR-605 variant by SNP-RFLP. Results: Overexpressing miR-605 in two TP53 mutated cell lines led to a significant attenuation in cell viability as measured by MTS assay. The capacity of these cell lines to form macroscopic colonies was also compromised following transfection by a miR-605 commercial mimic. Moreover, miR-605-transfected cells also displayed higher sensitivity to a wide range of chemotherapeutic drugs. In RD cells, MDM2 downregulation was detected following miR-605 overexpression but TP53 levels remained unchanged. Genotyping our aforementioned study population revealed the G-allele of the miR-605 SNP to be associated with an earlier age of cancer onset (p=0.02). The median age of cancer onset in carriers of the G-allele (n= 15) was 3.75 years compared to 21.5 years in patients with the common homozygous A/A genotype (n= 32). Conclusion: Together, our results identify miR-605 deregulation as a potential cancer modifying event in TP53 mutation carriers. Future work will focus on elucidating the functional consequences of the miR-605 SNP whilst also pursuing a more general understanding of miR-605 tumor suppression. Citation Format: Badr Idsaid, David Malkin. MicroRNA-605: A novel genetic modifier in Li-Fraumeni Syndrome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2013-3063

Comparison of southern Chinese Han and Brazilian Caucasian mutation rates at autosomal short tandem repeat loci used in human forensic genetics

The short tandem repeat (STR) loci used in human genetic studies are characterized by having relatively high mutation rates. In particular, to ensure an appropriate evaluation of genetic evidence in parentage and forensic analyses, it is essential to have accurate estimates of the mutation rates associated with the commonly used autosomal and sex chromosome STR loci. Differences in STR mutation rates between different ethnic groups should also be determined. Mutation data from two laboratories working with different ethnic groups were extracted from many meiotic transmissions ascertained for 15 autosomal STR loci currently used in forensic routine. Forty-five thousand and eighty-five trios were checked for the biological consistency of maternity and paternity through the analysis of a minimum of 15 loci. Mutations were scored as paternal, maternal, or ambiguous according to the most parsimonious explanation for the inconsistency, using always the least requiring hypothesis in terms of number of repeat differences. The main findings are: (a) the overall mutation rate across the 15 loci was 9.78 × 10(-4) per gamete per generation (95% CI = 9.30 × 10(-4)-1.03 × 10(-3)), and with just 48 (out of 1,587) exceptions, all of the mutations were single-step; (b) repeat gains were more frequent than losses; (c) longer alleles were found to be more mutable; and (d) the mutation rates differ at some loci between the two ethnic groups. Large worldwide meiotic transmission datasets are still needed to measure allele-specific mutation rates at the STR loci consensually used in forensic genetics.

Carrier Screening for Cystic Fibrosis among IVF Patients Utilizing Next Generation DNA Sequencing Detects Common, Rare, and Otherwise Undetectable Mutations

Carrier screening for cystic fibrosis (CF) is recommended by the American College of Medical Genetics, the American Congress of Obstetricians and Gynecologists and societies representing the Ashkenazi Jewish population. Due to cost considerations and restrictive technologies, traditional CF carrier screening assays are designed to look for only the most common CF mutations. Next generation DNA sequencing (NGS) allows for comprehensive determination of CF carrier status through its ability to detect a much larger set of mutations, thereby increasing detection of CF mutations in Caucasians, as well as in patients of other or mixed ethnicities. NGS is expected to increase the sensitivity of preconception screening for CF carriers over traditional assays by increasing dramatically the number of CF mutations detected (≥550 vs. ∼100). Our objective was to evaluate the clinical effectiveness of NGS for detecting not only common but also rare CF mutations that would go undetected using traditional carrier screening assays. A high-throughput and proprietary methodology (comprised of multiplex gene capture, NGS and computational analysis), which looks for over 550 known pathogenic CF mutations, was applied to over 4,000 patient DNA samples, as of this writing. Clinical reports were issued on the presence or absence of disease-causing CF mutations. Among 4,000 patients screened for CF utilizing NGS, we were able to detect 114 carriers, a frequency consistent with that reported in the literature given the ethnic mix tested. Several of these mutations – including S1455X (c.4364C>G), P67L (c.200C>T), Q1476X (c.4426C>T) - were not detected by traditional screening assays. Had traditional screening assays been used, these carriers would have been missed, thereby increasing the risk of having a child with CF. Additionally, during test validation, a DNA sample from a CF patient was found to have not only a known mutation (R334/c.100C>T) from one parent, but also a previously uncharacterized mutation (c.3368-2A>T) from the other parent. If the parent carrying c.3368-2A>T had been tested with a traditional, more limited carrier screening assay, this at-risk couple would have been missed. NGS has significant advantages over traditional carrier screening assays. Due to the vast number of pathogenic mutations detectable, NGS enables comprehensive examination of carrier status for CF in persons of all ethnicities, and is, therefore, expected to yield higher detection rates, resulting in fewer missed carriers.

MUTYH c.933+3A&gt;C, associated with a severely impaired gene expression, is the first Italian founder mutation in MUTYH‐Associated Polyposis

MUTYH variants are differently distributed in geographical areas of the world. In MUTYH-associated polyposis (MAP) patients from North-Eastern Italy, c.933+3A>C (IVS10+3A>C), a transversion causing an aberrant splicing process, accounts for nearly 1/5 of all mutations. The aim of this study was to verify whether its high frequency in North-Eastern Italy is due to a founder effect and to clarify its impact on MUTYH transcripts and protein. Haplotype analysis and age estimate performed on members of eleven Italian MAP families and cancer-free controls provided evidence that c.933+3A>C is a founder mutation originated about 83 generations ago. In addition, the Italian haplotype associated with the c.933+3A>C was also found in German families segregating the same mutation, indicating it had a common origin in Western Europe. Altogether c.933+3A>C and the two common Caucasian mutations p.Tyr179Cys and p.Gly396Asp represent about 60% of MUTYH alterations in MAP patients from North-Eastern Italy, suggesting the opportunity to perform targeted molecular screening for these variants in the diagnostic setting. Expression analyses performed on lymphoblastoid cell lines supported the notion that MUTYH c.933+3A>C alters splicing causing the synthesis of a non functional protein. However, some primary transcripts escape aberrant splicing, producing traces of full-length transcript and wild-type protein in a homozygote; this is in agreement with clinical findings that suggest a relatively mild phenotypic effect for this mutation. Overall, these data, that demonstrate a founder effect and further elucidate the splicing alterations caused by the MUTYH c.933+3A>C mutation, have important implications for genetic counseling and molecular diagnosis of MAP.